The correct timing and pattern of cell differentiation is essential to development of multicellular organisms, yet biologists understand little of how tissues and organs are specified at the correct time and synchronized with one another during development. We are exploiting the power of C. elegans genetics to identifY important temporal control genes as mutants that express cell fates (and hence, tissues) earlier or later than wild-type animals. These mutants define genes, termed heterochronic genes, that control the timing of development. In this proposal we will explore the mode of action of the lin-14 heterochronic gene. The level of LIN- 14 protein forms a temporal gradient that specifies the timing of stage-specific cell lineages. Mutations that perturb this level perturb the temporal sequence of cell lineages during development. LIN- 14 is a nuclear protein, but is not homologous to known proteins. Iherefore, the method by which graded LIN-14 levels act to specifY stage-specific cell fates, including the mechanism used by LIN-l4 to control downstream genes (i.e. transcription, splicing etc.) must be experimentally established. To do this, we have sought factors that mediate lin-14 action, such as lin-14 targets and combinatorial genes that act with lin-14. These have been identified genetically as suppressors or enhancers of Iin-14 mutant phenotypes. This proposal tests the hypothesis that these candidates define new genes involved in lin-14 control. The nature of these gene products is critical to understanding the mechanism of action of UN- 14. New genes will be cloned and ordered into the heterochronic pathway. The relationship between these new genes and lin-14 will be examined, e.g. does LIN-14 bind to the new gene products or regulate their expression? - We hypothesize that LIN- 14 acts with other proteins to control developmental timing. To identifY proteins that directly bind to the LIN- 14 protein, we have performed a yeast two hybrid screen and identified six genes whose products physically interact with LEN- 14. Preliminary data suggest that functional knock-out (by RNA interference) of two of these interactor genes leads to a heterochronic phenotype. One of these encodes a transcription factor, the other a 14-3-3 protein. The laboratory will test the hypothesis that these genes play an important role in Iin-14 control, by seeking or generating null mutations in these genes, and examining their effects. We will undertake a careful phenotypic analysis of all of the mutants isolated in this proposal, looking for heterochronic phenotypes, and for the potential of these mutations to suppress or enhance a lin-14 mutation. We also propose to examine the expression pattern of any new heterochronic genes in wild-type and lin-14 mutant backgrounds. Control of developmental timing is a fundamental problem faced by all animals. Since some genes in the heterochronic pathway have orthologues in mammals, the principles for control of developmental timing discovered in this work may be more general. Here, an understanding of heterochronic genes in a simple animal system, C. elegans, may offer a springboard for studies of temporal patterning in more complex organisms, including humans.